C.A.M.F. and L.S. participated in protocol design, acquisition and analysis of data, and drafting of the article. 
The authors wish to thank Dr. Kain, Dr. Loutfy, Dr. Wasmuth, and Dr. Ayi for their contributions.
C.F. was supported by a CTN/Ontario HIV Treatment Network (OHTN) postdoctoral fellowship. L.S. is supported by an OHTN Junior Investigator Development Award. The present work was supported by a Canadian Institutes of Health Research (CIHR) operating grant (MOP-13721 and 115160), and a CIHR New Investigator Catalyst grant.

<ol>
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The authors have nothing to disclose.

وقد تم تحسين بروتوكول لدينا من أجل دراسة أكثر واقعية المشارك العدوى بفيروس نقص المناعة البشرية الملاريا في المختبر. أولا، يتعين على كرات الدم الحمراء البشرية الطازجة ومصل للثقافة طفيلي الملاريا. وهذا أمر حيوي للحصول على صحة السكان من طفيليات الملاريا. لست] الطفيليات لا يمكن أن تكون بديلا للطفيليات حية كما إنتاج السيتوكينات هو أسرع بكثير ومكثفة عند استخدام يعيش P. المنجلية المصابة RBC (PfRBC) 17،18. وبالإضافة إلى ذلك، وتفعيل أنواع الخلايا مثل الخلايا القاتلة الطبيعية، يتطلب PfRBCs كلها، ثم لا يعمل بكفاءة مع لست] الطفيلي. قد يكون هذا بسبب الحاجة للاتصال المباشر بين PfRBC والكريات البيض أو قد يكون راجعا إلى الطبيعة غير المستقرة للبروابط المستمدة من الطفيليات التي تتفاعل مع مستقبلات سطح الخلية 18. يجب أيضا الثقافات الملاريا PfRBC تكون متزامنة جيدا (بروتوكول 4) قبل التجربة. PfRBCs متزامنة تحسين استنساخ البيانات التجريبية. على الرغم من أن هذا البروتوكول ديالكتبة استخدام مرحلة أتروفة PfRBC لثقافة مشتركة، فإنه يمكن بسهولة تعديل لدراسة مرحلة حلقة PfRBC. الكريات البيض البشرية يمكن أن يصاب بشكل مصطنع مع فيروس نقص المناعة البشرية، واستخدمت هذه الطريقة في الدراسات الملاريا نقص المناعة البشرية أبحاث العدوى شارك 20. ومع ذلك، فإن هذا لا نمذجة ديسريغولاتيون المناعي الذي ينجم عن الإصابة بفيروس نقص المناعة المزمن. في هذا النظام الثقافة المشتركة التي نستخدمها PBMCs معزولة من المشاركين البشري الذي مصابون بشكل مزمن مع HIV-1. عندما يطلب من المشاركين في الدراسة، من المهم تحديد هذه الفئة من السكان بعناية. معايير الانتقاء والإقصاء حيوية لضمان الحد الأدنى والحد الأقصى للتقلب التكاثر. وتضمنت معايير لدينا تعريف واضح للعدوى بفيروس نقص المناعة البشرية المزمن (HIV المصابة ل&gt; 1 سنة، مع CD4 + T-خلية تراجع عدد من&gt; 50 خلية / ملم 3 / سنة) واستبعاد أي شخص لديه عدوى متزامنة. تضمن هذا أن النتائج التي تم الحصول عليها يمكن أن يعزى ذلكليلي لتأثير الإصابة بفيروس نقص المناعة المزمن، وليس إصابة آخر. بالإضافة إلى ذلك، منذ كنا مهتمين الاستجابات المناعية الفطرية للملاريا استبعدنا الجهات المانحة التي كانت الإصابة بالملاريا السابق. وتستخدم الضوابط غير المصابة بفيروس نقص المناعة البشرية أيضا لكل فيروس نقص المناعة البشرية (+) المانحة، في كل نقطة زمنية أخذ العينات، للسماح للتطبيع البيانات. وينبغي بذل محاولة لمطابقة ضوابط لالمعني بفيروس نقص المناعة البشرية (+) المانحة من أجل سن على الأقل ولكن يفضل أن يكون أيضا الجنس (على الرغم من أن في دراستنا السابقة 12 كننا لم نلاحظ فرقا كبيرا في مجموعات فرعية خلية أو ردود خلوى بين الذكور والإناث بفيروس نقص المناعة البشرية المشاركين غير مصاب). إذا أخذ العينات المحتملين ومن المقرر الحفاظ على نفس السيطرة HIV-المعافين عن كل نقطة زمنية أخذ العينات هو مفيد. يمكن الاستجابات تختلف من تجربة إلى تجربة بسبب عوامل متعددة بما في ذلك صحة PfRBCs، درجة من التزامن، ومستوى الطفيل ومرحلة نضج PfRBCs، ومستوى الهيماتوكريت. رعايةيجب اتخاذها لإبقاء أكبر عدد ممكن من هذه متسقة بين التجارب. تطبيع إلى ضبط فيروس نقص المناعة البشرية غير مصاب يسمح لبعض المحاسبية لهذه المتغيرات. باستخدام خلايا جديدة أمر بالغ الأهمية لتجنب النتائج الاصطناعية. يمكن أن عينات تجميد والذوبان يكون لها تأثير كبير على بقاء الخلية 21،22، إنتاج السيتوكينات 23-26 والمظهرية سطح الخلية علامات 27. إذا حيدات ذات أهمية خاصة، فمن المهم أن الزجاج لا تستخدم خلال البروتوكول. حيدات تلتزم الزجاج والعديد من مواد بلاستيكية أخرى 28. نحن نستخدم مادة البولي بروبيلين الماصات البلاستيكية، ماصات نقل، وأنابيب في جميع أنحاء لتقليل الالتزام الوحيدات وإزالة نهائي من السكان الخلية دراستنا. عند إعداد التدفق الخلوي مقايسة، أي مزيج من الأجسام المضادة يمكن استخدامها تبعا للخلايا الفائدة. كنا مهتمون بشكل خاص في IFNγ وTNF الإنتاجمن الخلايا القاتلة الطبيعية والخلايا NKT وγδ الخلايا التائية. هذا تعريف لوحة الأجسام المضادة لدينا. ومع ذلك، إذا كان استخدام مجموعة مختلفة، فمن المهم لتحسين كميات من الأجسام المضادة لكل لوحة. وهذا أمر حيوي لتجنب بيانات خاطئة. أيضا، لتجنب التقلبات وضمان صحة، يطلب FMOs لتقييم مستويات خلوى الخلفية لكل حالة (RBC، PfRBC والمتوسطة، وسلطة النقد الفلسطينية / Ionomycin) وعدد المشتركين (HIV (-) وفيروس نقص المناعة البشرية (+)، الشكل 2). قياس في المختبر إنتاج السيتوكينات داخل الخلايا عادة ما يؤدي إلى مستويات خلفية عالية، وخصوصا عندما تم تربيتها خلايا قبل تلطيخ. منذ الشروط ثقافة تؤثر على مستويات الخلفية، FMOs مناسبة تضمن معرفة مستوى الخلفية لكل عينة. كان لدينا امدادات من الخلايا محدود، لذلك قمنا بتصميم FMOs لدينا لاحتواء كل البقع السطحية ولكن لا البقع داخل الخلايا. وهذا يسمح للمقارنة محددة من مستويات الخلفية خلوى على جميع أنواع مختلفة من الخلايا التي تمت دراستها.نحن أيضا ركض وصمة عار واحدة واحدة في التجربة لكل من علامات سطح لتأكيد مستويات خلفياتهم. بروتوكول صفها هو واحد تنوعا، والتي يمكن استخدامها لدراسة الردود في غضون ساعات أو أيام اعتمادا على خلايا الفائدة. لالأمثل إنتاج السيتوكينات PfRBC التي يسببها من الخلايا الليمفاوية الفطرية، قمنا بقياس التدفق الخلوي الانتاج بعد 2 أو 3 أيام. عند النظر في وحيدات، وينصح نقطة زمنية سابقة (1 أو 2 أيام). يسمح هذا النظام لأنواع متعددة الخلايا واستجابات الخلايا لتمييزها عن طريق التدفق الخلوي، ردود إفرازية إلى أن تقاس في supernatants الخلية، وملامح التعبير ينبغي تقييمها في استخراج الحمض النووي الريبي. وعلاوة على ذلك، واستخدام أجسام مضادة لعرقلة مستقبلات محددة أو تحييد يمكن استخدام السيتوكينات في هذا النظام لمزيد من تشريح الآليات. وقد استخدمنا بنجاح IL-18 مستقبلات الحصار لتوريط IL-18 مستقبلات في ردود IFNγ PfRBC التي يسببها 12. ويمثل هذا النظام واقعيطريقة جيم يمكن من خلالها تقييم عدد من الاستجابات المناعية الفطرية للملاريا في سياق الإصابة بالفيروس.

شارك العدوى، عدوى الالتهابات المتزامنة متعددة، هو القاعدة في البيئات الطبيعية. شارك في العدوى يمكن أن يكون لها تأثير كبير على علم الأمراض، وعلى التدبير العلاجي السريري لكل العدوى. في سياق التعاون العدوى، لقاح ونجاعة الأدوية، وكذلك الاختبارات التشخيصية، ويمكن أن يتأثر سلبا (إعادة النظر في 1). ومع ذلك، على الرغم من أهميته، فإن غالبية الأبحاث الممرض تعتبر التهابات واحدة فقط. الملاريا وHIV-1 (HIV) هي الأسباب الرئيسية للمراضة والوفيات على مستوى العالم. مناطق توطن الملاريا وفيروس نقص المناعة البشرية تشترك في تداخل جغرافي واسع، ووضع الملايين من الأشخاص المعرضين لخطر الاصابات المشتركة، وبالتالي لخطر المرض السريري أكثر شدة 2-10. هذين المرضين تتفاعل سلبا. في الأشخاص المصابين بفيروس نقص المناعة البشرية، وارتفاع الأحمال الفيروسية فيروس نقص المناعة البشرية والانخفاضات المؤقتة في CD4 + T التهم الخلايا يمكن أن ينظر إليها خلال عدوى الملاريا، في حينالملاريا طفيلي أعباء ومخاطر الملاريا السريرية وشديد أعلى في المصابين شارك أفراد 2،3،5،7،8،10. الآليات التي يزيد فيروس نقص المناعة البشرية شدة الملاريا ليست مفهومة تماما وتبرر اجراء مزيد من التحقيقات. نحن هنا وصف الطريقة التي الملاريا وشارك في الإصابة بفيروس نقص المناعة يمكن دراستها في المختبر وعلى وجه التحديد، ويسمح هذا الأسلوب لفحص ردود الملاريا محددة المناعية في سياق الإصابة بالفيروس. يصف بروتوكول لدينا نظام متعدد الاستعمالات شارك في الثقافة باستخدام خلايا الدم وحيدات النوى الطرفية المعزولة حديثا (PBMCs) معزولة عن مزمنة بفيروس نقص المناعة البشرية المانحين المصابة في المختبر مثقف P. مطفول المنجلية كريات الدم الحمراء (PfRBC). ويمكن أيضا أن تدرس تأثير العلاج المضاد للفيروس نقص المناعة البشرية على هذه الردود باستخدام PBMCs جمعت بأثر رجعي من فيروس نقص المناعة البشرية (+) الجهات المانحة قبل وبعد العلاج. وقد استخدمنا هذا النظام للتحقيق في تأثير فيروس نقص المناعة البشريةعلى الاستجابات المناعية الفطرية الملاريا محددة 11،12، وتمكنوا من تحديد أن IFNγ وTNF الردود الملاريا محددة يعانون من ضعف في الخلايا القاتلة الطبيعية والخلايا NKT، γδ الخلايا التائية من فيروس نقص المناعة البشرية (+) الجهات المانحة قبل وبعد العلاج المضاد للفيروس نقص المناعة البشرية . بالإضافة إلى ذلك، كنا قادرين على استخدام هذا النظام لتحديد أن ظائف الوحيدات يتم تخفيض قيمة أيضا في فيروس نقص المناعة البشرية (+) الجهات المانحة، ولكن التعافي بعد فيروس نقص المناعة البشرية العلاج المضاد للفيروسات الرجعية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>alanine</td>
			<td>Sigma</td>
			<td>A7377</td>
			<td></td>
		</tr>
		<tr>
			<td>antibodies (see other table)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm with BD GolgiPlug</td>
			<td>BD</td>
			<td>555028</td>
			<td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td>
		</tr>
		<tr>
			<td>BD Vacutainer ACD Solution A</td>
			<td>BD</td>
			<td>364506</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Sodium Heparin</td>
			<td>BD</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no calcium, no magnesium)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>heat inactivate before use</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin (10mg/ml)</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hema3 Staining Set</td>
			<td>Fisher</td>
			<td>122-911</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxanthine</td>
			<td>Sigma</td>
			<td>H9636</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I3909</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (10mM)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 powder</td>
			<td>Life-Technologies</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 with L-glutamine and HEPES</td>
			<td>Thermo Scientific</td>
			<td>SH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate (powder, cell culture)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100mM)</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Trizma base)</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Ambion</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>BD CompBead</td>
			<td>BD</td>
			<td>552843, 552845</td>
			<td>depends on antibodies used</td>
		</tr>
		<tr>
			<td>Parasite Gas Mixture</td>
			<td>By special order</td>
			<td></td>
			<td>3% CO2, 1% O2, balance N2</td>
		</tr>
	</tbody>
</table>

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

الرسوم البيانية تصور مستويات الإنتاج IFNγ من خلايا NKT (الشكل 2)، وذلك باستخدام CD56 + CD3 + γδ- بوابات للحصول على السكان الخلايا NKT (لا تظهر البيانات). كان المثقف الخلايا لمدة 72 ساعة قبل التلوين. مرة واحدة الملون، تم الحصول على 100،000 CD3 + الخلايا على قياس التدفق الخلوي للحصول على عدد كبير من السكان ما يكفي من NK، NKT وخلايا γδ (خلايا الفائدة). يتم عرض ما لا يقل عن 5600 الخلايا NKT على كل رسم بياني. يتم الحصول على إنتاج TNF بنفس الطريقة (لا تظهر البيانات). تظهر الرسوم البيانية بوضوح أن إنتاج IFNγ أقل في الخلايا من فيروس نقص المناعة البشرية (+) الأفراد مقارنة مع فيروس نقص المناعة البشرية (-) أشخاص المعرضين لPfRBC. تحليل التدفق الخلوي هو شخصي جدا. ولذلك فمن الضروري أن يكون كل الضوابط المناسبة لكل تجربة (انظر الشكل 2). وتحسب مستويات الخلفية باستخدام عينات FMO، والذي يسمح للتمثيل الحقيقي للتلطيخ خلوى.استخدمت خلايا حفز مع سلطة النقد الفلسطينية / Ionomycin كعنصر تحكم إيجابية (لا تظهر البيانات). سلطة النقد الفلسطينية وIonomycin هي محفزات قوية للT إنتاج السيتوكينات الخلية. وعدم وجود إنتاج IFNγ في هذه العينات من المرجح أن تشير إلى وجود مشكلة مع بروتوكول تلطيخ. ومع ذلك، متغيرات أخرى مثل بقاء الخلية أو الكواشف الخاملة قد يكون أيضا على خطأ. <img alt="الشكل 1" src="/files/ftp_upload/52969/52969fig1.jpg" /> الشكل 1. تصوير من Ficoll آخر التدرج تدور يدل على موقف PBMCs. <img alt="الرقم 2" src="/files/ftp_upload/52969/52969fig2.jpg" /> الشكل 2. إنتاج الإنترفيرون γ الخلايا الطبيعية القاتلة T. تم الحصول على مخططات تدفق التي تبوب على CD56 + CD3 + γδ- خلايا (لا تقل عن 5600 الأحداث). إنتاج IFNγ غير قابلة للكشف في فيروس نقص المناعة البشرية (-) عينة حفز معالمتصورة المنجلية المصابة خلايا الدم الحمراء. هذا الرد خلوى لم يعد واضحا في سياق الإصابة بفيروس نقص المناعة المزمن. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52969/52969fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

ل<em&gt; في المختبر</em&gt; نموذج لقياس الاستجابات المناعية لمكافحة الملاريا في سياق الفيروس المسبب للإيدز

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

an-vitro-model-for-measuring-immune-responses-to-malaria-context-hiv

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

10.2K Views.  University of Calgary. Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection. 

Video: An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

نموذج مثلي من سرطان المبيض في الفئران مصلي مناعيا ل في الجسم الحي التصوير الورم ورصد الاستجابات المناعية ورم

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

الاستدلال الوراثي من HIV - 1 Tropism باستخدام السكان على أساس تسلسل V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

سبع خطوات لخلايا النجمية

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

قياس الحمولة الجرثومية والاستجابات المناعية في الفئران المصابة المستوحدة الليستيريا</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

Uncoating في المختبر من HIV - 1 النوى

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

و<em&gt; في المختبر</em&gt; المشارك عدوى نموذج لدراسة<em&gt; المتصورة المنجلية</em&gt; HIV-1-التفاعلات في الإنسان خلايا الوحيدات المشتقة الابتدائية المناعي

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

التحقيق في آثار البروبيوتيك على المكورات الرئوية الاستعمار باستخدام<em&gt; في المختبر</em&gt; الالتزام الفحص

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

في تحليل المختبر من الاستجابة المناعية الخلوية بوساطة Myd88 لفيروس غرب النيل المسخ سلالة العدوى

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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نموذج مثلي من سرطان المبيض في الفئران مصلي مناعيا ل في الجسم الحي التصوير الورم ورصد الاستجابات المناعية ورم

الاستدلال الوراثي من HIV - 1 Tropism باستخدام السكان على أساس تسلسل V3

سبع خطوات لخلايا النجمية

قياس الحمولة الجرثومية والاستجابات المناعية في الفئران المصابة المستوحدة الليستيريا

Uncoating في المختبر من HIV - 1 النوى

و

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

التحقيق في آثار البروبيوتيك على المكورات الرئوية الاستعمار باستخدام

في تحليل المختبر من الاستجابة المناعية الخلوية بوساطة Myd88 لفيروس غرب النيل المسخ سلالة العدوى

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